Xenograft heart valves

ABSTRACT

The invention provides an article of manufacture comprising a substantially non-immunogenic heart valve xenograft for implantation into humans. The invention further provides methods for preparing a heart valve xenograft by removing at least a portion of a soft tissue from a non-human animal to provide a xenograft; washing the xenograft in saline and alcohol; subjecting the xenograft to cellular disruption treatment; treating the xenograft with crosslinking agents, and digesting the xenograft with a proteoglycan-depleting factor and/or glycosidase. The invention also provides an article of manufacture produced by the above-identified method of the invention. The invention further provides a heart valve xenograft for implantation into a human including a portion of a heart valve from a non-human animal, wherein the portion has extracellular components and substantially only dead cells. The extracellular components have reduced proteoglycan molecules. Each of the xenografts of the invention are substantially non-immunogenic and have substantially the same mechanical properties as a corresponding native heart valve.

This application is a continuation application of U.S. application Ser.No. 10/139,499, filed on May 6, 2002, now abandoned, which claimspriority to U.S. application Ser. No. 09/585,509, filed on Jun. 1, 2000,the contents of all of which are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to the field of treatment of defectivehuman heart valves, and in particular, to replacement and repair ofdefective or damaged human heart valves using a substantiallyimmunologically compatible heart valve from a non-human animal.

BACKGROUND OF THE INVENTION

Heart valves are composed of fibrochondrocytes and an extracellularmatrix of collagen and elastic fibers, as well as a variety ofproteoglycans. Various synthetic and tissue based materials (the lattereither from the recipient organism or from a different organism withinthe same species) have been used for forming heart valve replacements.Each have their advantages and disadvantages.

In the case of synthetic heart valves, it may be possible to modifyadvantageously the properties of the heart valves by altering themonomers and/or the reaction conditions of the synthetic polymers.Synthetic heart valves may be associated with thromboembolism andmechanical failure, however. See U.S. Pat. No. 4,755,593.

Tissue based heart valves may demonstrate superior blood contactingproperties relative to their synthetic counterparts. Tissue based heartvalves also may be associated with inferior in vivo stability, however.See U.S. Pat. No. 4,755,593.

Pericardial xenograft tissue valves have been introduced as alternativesto the synthetic and the tissue based valves described above. SeeIonescu, M. I. et al., Heart Valve Replacement With The Ionescu-ShileyPericardial Xenograft, J. Thorac. Cardiovas. Surg. 73; 31-42 (1977).Such valves may continue to have calcification and durability problems,however. See Morse, D, ed. Guide To Prosthetic Heart Valves,Springer-Verlag, New York, 225-232 (1985).

Accordingly, there is a need for mechanically durable, flexible heartvalves replacements which are capable of contacting the blood and arestable in vivo.

Much of the structure and many of the properties of original heartvalves may be retained in transplants through use of heterograft orxenograft materials, that is, heart valve from a different species thanthe graft recipient. For example, tendons or ligaments from cows orother animals are covered with a synthetic mesh and transplanted into aheterologous host in U.S. Pat. No. 4,400,833. Flat tissues such as pigpericardia are also disclosed as being suitable for heterologoustransplantation in U.S. Pat. No. 4,400,833. Bovine peritoneum fabricatedinto a biomaterial suitable for prosthetic heart valves, vasculargrafts, burn and other wound dressings is disclosed in U.S. Pat. No.4,755,593. Bovine, ovine, or porcine blood vessel xenografts aredisclosed in WO 84/03036. However, none of these disclosures describethe use of a xenograft for heart valve replacement.

Once implanted in an individual, a xenograft provokes immunogenicreactions such as chronic and hyperacute rejection of the xenograft. Theterm “chronic rejection”, as used herein, refers to an immunologicalreaction in an individual against a xenograft being implanted into theindividual. Typically, chronic rejection is mediated by the interactionof IgG natural antibodies in the serum of the individual receiving thexenograft and carbohydrate moieties expressed on cells, and/or cellularmatrices and/or extracellular components of the xenograft. For example,transplantation of heart valve xenografts from nonprimate mammals (e.g.,porcine or bovine origin) into humans is primarily prevented by theinteraction between the IgG natural anti-Gal antibody present in theserum of humans with the carbohydrate structure Galα1-3Galβ1-4GlcNAc-R(α-galactosyl or α-gal epitope) expressed in the xenograft. K. R. Stoneet al., Porcine and bovine cartilage transplants in cynomolgus monkey:I. A model for chronic xenograft rejection, 63 Transplantation 640-645(1997); U. Galili et al., Porcine and bovine cartilage transplants incynomolgus monkey: II. Changes in anti-Gal response during chronicrejection, 63 Transplantation 646-651 (1997). In chronic rejection, theimmune system typically responds within one to two weeks of implantationof the xenograft.

In contrast with “chronic rejection”, “hyperacute rejection” as usedherein, refers to the immunological reaction in an individual against axenograft being implanted into the individual, where the rejection istypically mediated by the interaction of IgM natural antibodies in theserum of the individual receiving the xenograft and carbohydratemoieties expressed on cells. This interaction activates the complementsystem, causing lysis of the vascular bed and stoppage of blood flow inthe receiving individual within minutes to two to three hours.

The term “extracellular components”, as used herein, refers to anyextracellular water, collagen and elastic fibers, proteoglycans,fibronectin, elastin, and other glycoproteins, which are present inheart valve.

Xenograft materials may be chemically treated to reduce immunogenicityprior to implantation into a recipient. For example, glutaraldehyde isused to cross-link or “tan” xenograft tissue in order to reduce itsantigenicity, as described in detail in U.S. Pat. No. 4,755,593. Otheragents such as aliphatic and aromatic diamine compounds may provideadditional crosslinking through the side chain carboxyl groups ofaspartic and glutamic acid residues of the collagen polypeptide.Glutaraldehyde and diamine tanning also increases the stability of thexenograft tissue.

Xenograft tissues may also be subjected to various physical treatmentsin preparation for implantation. For example, U.S. Pat. No. 4,755,593discloses subjecting xenograft tissue to mechanical strain by stretchingto produce a thinner and stiffer biomaterial for grafting. Tissue forallograft transplantation is commonly cryopreserved to optimize cellviability during storage, as disclosed, for example, in U.S. Pat. Nos.5,071,741; 5,131,850; 5,160,313; and 5,171,660. U.S. Pat. No. 5,071,741discloses that freezing tissues causes mechanical injuries to cellstherein because of extracellular or intracellular ice crystal formationand osmotic dehydration.

SUMMARY OF THE INVENTION

The present invention provides a substantially non-immunogenic heartvalve xenograft for implantation into a human in need of heart valverepair or replacement. The invention further provides methods forprocessing xenogeneic heart valve with reduced immunogenicity but withsubstantially native elasticity and load-bearing capabilities forxenografting into humans.

As used herein, the term “xenograft” is synonymous with the term“heterograft” and refers to a graft transferred from an animal of onespecies to one of another species. Stedman's Medical Dictionary,Williams & Wilkins, Baltimore, Md. (1995).

As used herein, the term “xenogeneic”, as in, for example, xenogeneicheart valve, refers to heart valve transferred from an animal of onespecies to one of another species. Id.

The methods of the invention, include, alone or in combination,treatment with radiation, one or more cycles of freezing and thawing,treatment with a chemical cross-linking agent, treatment with alcohol orozonation, and sterilization In addition to or in lieu of these methods,the methods of the invention include, alone or in combination, in anyorder, a cellular disruption treatment, glycosidase digestion ofcarbohydrate moieties of the xenograft, or treatment withproteoglycan-depleting factors. Optionally, the xenograft can be exposedto an aldehyde for further crosslinking. After one or more of theabove-described processing steps, the methods of the invention provide axenograft having substantially the same mechanical properties as anative heart valve.

As used herein, the term “cellular disruption” as in, for example,cellular disruption treatment, refers to a treatment for killing cells.

In one embodiment, the invention provides an article of manufacturecomprising a substantially non-immunogenic heart valve xenograft forimplantation into a human.

In another embodiment, the invention provides a method of preparing aheart valve xenograft for implantation into a human, which includesremoving at least a portion of a heart valve from a non-human animal toprovide a xenograft; washing the xenograft in water and alcohol; andsubjecting the xenograft to at least one treatment selected from thegroup consisting of exposure to ultraviolet radiation, immersion inalcohol, ozonation, and freeze/thaw cycling, whereby the xenograft hassubstantially the same mechanical properties as a corresponding portionof a native heart valve.

In yet still a further embodiment, the invention provides a xenograftformed of a soft tissue for implantation into a human comprising aportion of the soft tissue from a nonhuman animal, wherein the portionincludes a plurality of extracellular components, a plurality ofsubstantially only dead cells, and an aldehyde in an amount ranging fromabout 0.01% to about 5% crosslinking a plurality of proteins of theextracellular components, the extracellular components and the deadcells having substantially no surface carbohydrate moieties which aresusceptible to glycosidase digestion, and whereby the portion issubstantially non-immunogenic and has substantially the same mechanicalproperties as a corresponding portion of the native soft tissue, andwherein the soft tissue is suitable for use as heart valve xenograftmaterial.

As used herein, the term “portion” refers to all or less than all of therespective soft tissue heart valve xenograft material. “Soft tissuexenograft material” refers to the non-human heart valves, valveportions, such as leaftlets, and other soft tissue materials that can befashioned into valves and valve portions, such as, for example,pericardium.

In another embodiment, the invention provides a method of preparing aheart valve xenograft for implantation into a human, which includesremoving at least a portion of a heart valve from a non-human animal toprovide a xenograft; washing the xenograft in water and alcohol;subjecting the xenograft to a cellular disruption treatment; anddigesting the xenograft with a glycosidase to remove surfacecarbohydrate moieties, whereby the xenograft has substantially the sameproperties as a corresponding portion of a native heart valve. As usedherein, the term “surface carbohydrate moiety (moieties)” refers to aterminal α-galactosyl sugar at the non-reducing end of a carbohydratechain.

In a further embodiment, the invention provides a method of preparing aheart valve xenograft for implantation into a human, which includesremoving at least a portion of heart valve from a non-human animal toprovide a xenograft; washing the xenograft in water and alcohol;subjecting the xenograft to a cellular disruption treatment; anddigesting the xenograft with a proteoglycan-depleting factor to removeat least a portion of the proteoglycans from the xenograft, whereby thexenograft has substantially the same mechanical properties as acorresponding portion of a native heart valve and is substantiallynon-immunogenic.

In yet further embodiments, the invention provides articles ofmanufacture including substantially non-immunogenic heart valvexenografts for implantation into humans produced by one or more of theabove-identified methods of the invention.

In another embodiment, the invention provides a heart valve xenograftfor implantation into a human which includes a portion of a heart valvefrom a non-human animal, wherein the portion has substantially nosurface carbohydrate moieties which are susceptible to glycosidasedigestion, and whereby the portion has substantially the same mechanicalproperties as a corresponding portion of a native heart valve. In stillyet another embodiment, the invention provides a heart valve xenograftfor implantation into a human which includes a portion of a heart valvefrom a non-human animal, wherein the portion includes extracellularcomponents and substantially only dead cells, the extracellularcomponents having reduced proteoglycans. The portion of the heart valveis substantially non-immunogenic and has substantially the samemechanical properties as the native heart valve.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed against the chronic rejection ofxenografts for implantation into humans. Accordingly, heart valvexenografts produced in accordance with the methods of the invention aresubstantially non-immunogenic, while generally maintaining themechanical properties of a native heart valve.

While the heart valve may undergo some shrinkage during processing, aheart valve xenograft prepared in accordance with the invention willhave the general appearance of a native heart valve xenograft. Forexample, a mitral valve xenograft prepared in accordance with theinvention will have the general appearance of a native mitral valve, andsemi-lunar valve xenografts of the invention will have the generalappearance of a native semi-lunar valves. The heart valve xenograft maybe valve segments, such as individual leaflets, each of which may beimplanted into receipient heart.

The invention provides, in one embodiment, a method for preparing orprocessing a xenogeneic heart valve for engraftment into humans. Theheart valve may be harvested from any non-human animal to prepare thexenografts of the invention. Heart valve from transgenic non-humananimals or from genetically altered non-human animals may also be usedas xenografts in accordance with the present invention. Preferably,bovine, ovine, or porcine hearts, and more preferably porcine hearts,serve as sources of heart valves used to prepare the xenografts.Alternatively, porcine pericardium can be used to form the heart valvexenografts of the present invention.

In the first step of the method of the invention, an intact heart isremoved from a non-human animal. Suitable heart valve tissues areexcised from the heart. Pericardium may be also harvested and implantedto replace or repair damaged heart valves by those of skill in the artusing known techniques. Preferably heart valve from a correspondingheart valve is used to make the heart valve xenograft of the invention.For example, mitral valve may be used to make a mitral valve xenograftfor implantation.

In accordance with the invention, the heart which serves as the sourceof the heart valve is collected from freshly killed animals andpreferably immediately placed in a suitable sterile isotonic or othertissue preserving solution. Preferably, harvesting of the hearts andvalves occurs as soon as possible after slaughter of the animal andpreferably is performed in the cold, i.e., in the approximate range ofabout 5° C. to about 20° C., to minimize enzymatic degradation of theheart valve, under strict sterile technique.

The harvested valves and tissue are dissected free of adjoining tissue.Alternatively, a valve may be dissected with portions of the surroundingcardiac tissue. For example, tricuspid valves are excised as separateleaflets, or as an intact valve including the fibrous ring surroundingthe auriculo-ventricular orifice and the tendinous chords. Once removed,optionally, the valve or valve portions are supported with stents, ringsand the like. The heart valve or portion is carefully identified anddissected free of adhering tissue, plaques, calcifications and the like,thereby forming the xenograft.

In one form of the invention, porcine peritoneum or pericardium isharvested to form a heart valve xenografts according to procedures knownto those of ordinary skill in the art. See, for example, the peritoneumharvesting procedure discussed in U.S. Pat. No. 4,755,593 by Lauren.

In a preferred form of the invention, the xenograft is then washed inabout ten volumes of sterile cold water to remove residual bloodproteins and water soluble materials. The xenograft is then immersed inalcohol at room temperature for about five minutes, to sterilize thetissue and to remove non-collagenous materials. After alcohol immersion,the xenograft may be directly implanted or may be subjected to at leastone of the following treatments: radiation treatment, treatment withalcohol, ozonation, one or more cycles of freezing and thawing, and/ortreatment with a chemical cross-linking agent. When more than one ofthese treatments is applied to the xenograft, the treatments may occurin any order.

In one embodiment of the method of the invention, the xenograft istreated by exposure to ultraviolet radiation for about fifteen minutesor gamma radiation in an amount of about 0.5 to 3 MegaRad.

In another embodiment, the xenogrart is treated by again being placed inan alcohol solution. Any alcohol solution may be used to perform thistreatment. Preferably, the xenograft is placed in a 70% solution ofisopropanol at room temperature.

In still another embodiment, the xenograft is subjected to ozonation.

In a further embodiment of the method of the invention, the xenograft istreated by freeze/thaw cycling. For example, the xenograft may be frozenusing any method of freezing, so long as the xenograft is completelyfrozen, i.e., no interior warm spots remain which contain unfrozen heartvalve tissue. Preferably, the xenograft is dipped into liquid nitrogenfor about five minutes to perform this step of the method. Morepreferably, the xenograft is frozen slowly by placing it in a freezer.In the next step of the freeze/thaw cycling treatment, the xenograft isthawed by immersion in an isotonic saline bath at room temperature(about 25° C.) for about ten minutes. No external heat or radiationsource is used, in order to minimize fiber degradation.

In yet a further embodiment, the xenograft optionally is exposed to achemical agent to tan or crosslink the proteins within the extracellularcomponents, to further diminish or reduce the immunogenic determinantspresent in the xenograft. Any tanning or crosslinking agent may be usedfor this treatment, and more than one crosslinking step may be performedor more than one crosslinking agent may be used in order to ensurecomplete crosslinking and thus optimally reduce the immunogenicity ofthe xenograft. For example, aldehydes such as glutaraldehyde,formaldehyde, adipic dialdehyde, and the like, may be used to crosslinkthe extracellular collagen of the xenograft in accordance with themethod of the invention. Other suitable crosslinking agents includealiphatic and aromatic diamines, carbodiimides, diisocyanates, and thelike.

When an aldehyde such as, for example, glutaraldehyde is used as thecrosslinking agent, the xenograft may be placed in a buffered solutioncontaining about 0.001% to about 5.0% glutaraldehyde and preferably,about 0.01% to about 5.0% glutaraldehyde, and having a pH of about 7.4.More preferably about 0.01% to about 0.10% aldehyde, and most preferablyabout 0.01% to about 0.05% aldehyde is used. Any suitable buffer may beused, such as phosphate buffered saline ortrishydroxymethylaminomethane, and the like, so long as it is possibleto maintain control over the pH of the solution for the duration of thecrosslinking reaction, which may be from one to fourteen days, andpreferably from one to five days, and most preferably from three to fivedays.

Alternatively, the xenograft can be exposed to a crosslinking agent in avapor form, including, but not limited to, a vaporized aldehydecrosslinking agent, such as, for example, vaporized formaldehyde. Thevaporized crosslinking agent can have a concentration and a pH and thexenograft can be exposed to the vaporized crosslinking agent for aperiod of time suitable to permit the crosslinking reaction to occur.For example, the xenograft can be exposed to vaporized crosslinkingagent having a concentration of about 0.001% to about 5.0% andpreferably, about 0.01% to about 5.0%, and a pH of about 7.4. Morepreferably, the xenograft is exposed to the aldehyde in an amountranging from about 0.01% to about 0.10%, and most preferably to analdehyde ranging in an amount from about 0.01% to about 0.05%. Thexenograft is exposed to the aldehyde for a period of time which can befrom one to fourteen days, and preferably from one to five days, andmost preferably from three to five days. Exposure to vaporizedcrosslinking agent can result in reduced residual chemicals in thexenograft from the crosslinking agent exposure.

The crosslinking reaction continues until the immunogenic determinantsare substantially eliminated from the xenogeneic heart valve, but thereaction is terminated prior to significant alterations of themechanical properties of the xenograft. When diamines are also used ascrosslinking agents, the glutaraldehyde crosslinking occurs after thediamine crosslinking, so that any unreacted diamines are capped. Afterthe crosslinking reactions have proceeded to completion as describedabove, the xenograft is rinsed to remove residual chemicals, and0.01-0.10 M glycine, and preferably, 0.01-0.05 M glycine is added to capany unreacted aldehyde groups which remain.

In addition to the above treatments, the xenograft is subjected to acellular disruption treatment to kill the xenograft's cells. Thecellular disruption treatment precedes or follows digestion of thexenograft with glycosidases to remove surface carbohydrate moieties fromthe xenograft. In addition or in lieu of the glycosidase treatment,either preceding or following the glycosidase treatment, the xenograftmay be treated with proteoglycan-depleting factors.

The xenograft is subjected to a cellular disruption treatment to killthe cells of the heart valve. Typically after surface carbohydratemoieties have been removed from living cells and the extracellularcomponents, the living cells reexpress the surface carbohydratemoieties. Reexpression of antigenic moieties of a xenograft can provokecontinued immunogenic rejection of the xenograft. In contrast, deadcells are unable to reexpress surface carbohydrate moieties. Removal ofantigenic surface carbohydrate moieties from dead cells and theextracellular components of a xenograft substantially permanentlyeliminates antigenic surface carbohydrate moieties as a source ofimmunogenic rejection of the xenograft.

Accordingly, in the above-identified embodiments, the xenograft of thepresent invention is subjected to freeze/thaw cycling as discussed aboveto disrupt, i.e., to kill the cells of the heart valve. Alternatively,the xenograft of the present invention is treated with gamma radiationhaving an amount of 0.2 MegaRad up to about 3 MegaRad. Such radiationkills the heart valve cells and sterilizes the xenograft. Once killed,the heart valve cells are no longer able to reexpress antigenic surfacecarbohydrate moieties such α-gal epitopes which are factors in theimmunogenic rejection of the transplanted xenografts.

Either before or after the heart valve cells are killed, in embodimentsof the invention, the xenograft is subjected to in vitro digestion ofthe xenograft with glycosidases, and specifically galactosidases, suchas α-galactosidase, to enzymatically eliminate antigenic surfacecarbohydrate moieties. In particular, α-gal epitopes are eliminated byenzymatic treatment with α-galactosidases, as shown in the followingreaction:

The N-acetyllactosamine residues are epitopes that are normallyexpressed on human and mammalian cells and thus are not immunogenic. Thein vitro digestion of the xenograft with glycosidases is accomplished byvarious methods. For example, the xenograft can be soaked or incubatedin a buffer solution containing glycosidase. In addition, the xenograftcan be pierced to increase permeability, as further described below.Alternatively, a buffer solution containing the glycosidase can beforced under pressure into the xenograft via a pulsatile lavage process.

Elimination of the α-gal epitopes from the xenograft diminishes theimmune response against the xenograft. The α-gal epitope is expressed innonprimate mammals and in New World monkeys (monkeys of South America)as 1×10⁶-35×10⁶ epitopes per cell, as well as on macromolecules such asproteoglycans of the extracellular components. U. Galili et al., Man,apes, and Old World monkeys differ from other mammals in the expressionof α-galactosyl epitopes on nucleated cells, 263 J. Biol. Chem. 17755(1988). This epitope is absent in Old World primates (monkeys of Asiaand Africa and apes) and humans, however. Id. Anti-Gal is produced inhumans and primates as a result of an immune response to α-gal epitopecarbohydrate structures on gastrointestinal bacteria. U. Galili et al.,Interaction between human natural anti-α-galactosyl immunoglobulin G andbacteria of the human flora, 56 Infect. Immun. 1730 (1988); R. M.Hamadeh et al., Human natural anti-Gal IgG regulates alternativecomplement pathway activation on bacterial surfaces, 89 J. Clin. Invest.1223 (1992). Since nonprimate mammals produce α-gal epitopes,xenotransplantation of xenografts from these mammals into primatesresults in rejection because of primate anti-Gal binding to theseepitopes on the xenograft. The binding results in the destruction of thexenograft by complement fixation and by antibody dependent cellcytotoxicity. U. Galili et al., Interaction of the natural anti-Galantibody with α-galactosyl epitopes: A major obstacle forxenotransplantation in humans, 14 Immunology Today 480 (1993); M.Sandrin et al., Anti-pig IgM antibodies in human serum reactpredominantly with Galα1-3Gal epitopes, 90 Proc. Natl. Acad. Sci. USA11391 (1993); H. Good et al., Identification of carbohydrate structureswhich bind human anti-porcine antibodies: implications for discordantgrafting in man. 24 Transplant. Proc. 559 (1992); B. H. Collins et al.,Cardiac xenografts between primate species provide evidence for theimportance of the α-galactosyl determinant in hyperacute rejection, 154J. Immunol. 5500 (1995). Furthermore, xenotransplantation results inmajor activation of the immune system to produce increased amounts ofhigh affinity anti-Gal. In accordance with the invention, thesubstantial elimination of α-gal epitopes from cells and fromextracellular components of the xenograft, and the prevention ofreexpression of cellular α-gal epitopes diminish the immune responseagainst the xenograft associated with anti-Gal antibody binding withα-gal epitopes.

In addition, the heart valve xenografts of the invention may be treatedwith polyethylene glycol (PEG) prior to or concurrently with treatmentwith glycosidase. PEG acts as a carrier for the glycosidase bycovalently bonding to the enzyme and to the collagen extracellularcomponents. Further, PEG-treated xenografts reduce immunogenicity.

Either before or after the xenograft cells are killed, in embodiments ofthe invention, the xenograft is washed or digested with one or moredifferent types of proteoglycan-depleting factors. Theproteoglycan-depleting factor treatment can precede or followglycosidase treatment. Proteoglycans such as glycosaminoglycans (GAGs)are interspersed either uniformly as individual molecules or withinvarying amounts within the extracellular components of the presentinvention's xenograft. The GAGs include mucopolysaccharide moleculessuch as chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate,dermatan sulfate, heparin sulfate, hyaluronic acid, and mixturesthereof. The proteoglycans including such GAGs contain attachedcarbohydrates such as α-gal epitopes. Such epitopes stimulate an immuneresponse once the xenograft is transplanted, as discussed above. Washingor digesting the xenograft with the proteoglycan-depleting factorremoves at least a portion of the proteoglycans and attached α-galepitopes from the extracellular components of the xenograft, and therebydiminishes the immune response against the xenograft upon itstransplantation. After the proteoglycan-depleting factor treatment andsubsequent transplantation, natural tissue repopulates the remainingcollagen shell.

Non-limiting examples of the proteoglycan-depleting factors used in thepresent invention include proteoglycan-depleting factors such aschondroitinase ABC, hyaluronidase, chondroitin AC II lyase, keratanase,trypsin, fibrinectin and fragments of fibronectin.

Other proteoglycan-depleting factors known to those of ordinary skill inthe art are also possible for use with the present invention, however.The present invention's xenograft is treated with proteoglycan-depletingfactor in an amount effective for removing at least a portion of theproteoglycans from the extracellular components of the xenograft.Preferably, the xenograft is treated with proteoglycan-depleting factorsuch as hyaluronidase in an amount ranging from about 1.0 TRU/ml toabout 100.0 TRU/ml or proteoglycan-epleting factor such aschondroitinase ABC in an amount ranging from about 0.01 u/ml to about2.0 u/ml or most preferably, in an amount ranging from about 1.0 ul/mlto about 2.0 u/ml. The xenograft can also be treated withproteoglycan-depleting factor such as fibronectin fragment, (e.g., aminoterminal 29-kDa fibronectin fragment) in an amount ranging from about0.1 μM to about 1.0 μM, and preferably in an amount ranging from about0.1 μM to about 1.0 μM.

Prior to treatment, the xenograft optionally may be pierced to increasepermeability to agents used to render the xenograft substantiallynon-immunogenic. A sterile surgical needle such as an 18 gauge needle isused to perform this piercing step, or, alternatively a comb-likeapparatus containing a plurality of needles are used. The piercing maybe performed with various patterns, and with various pierce-to-piercespacings, in order to establish a desired access to the interior of thexenograft. Piercing may also be performed with a laser. In one form ofthe invention, one or more straight lines of punctures about threemillimeters apart are established circumferentially in the surface ofthe xenograft.

Prior to implantation, the heart valve xenograft of the invention may betreated with limited digestion by proteolytic enzymes such as ficin ortrypsin to increase tissue flexibility, or coated with anticalcificationagents, antithrombotic coatings, antibiotics, growth factors, or otherdrugs which may enhance the incorporation of the xenograft into therecipient. The heart valve xenograft of the invention may be furthersterilized using known methods, for example, with additionalglutaraldehyde or formaldehyde treatment, ethylene oxide sterilization,propylene oxide sterilization, or the like. The xenograft may be storedfrozen until required for use.

The heart valve xenograft of the invention, or a segment thereof, may beimplanted into damaged human hearts by those of skill in the art usingknown surgical techniques, for example, by open heart surgery, orminimally invasive techniques such as endoscopic surgery, andtransluminal implantation. Specific instruments for performing suchsurgical techniques are known to those of skill in the art, which ensureaccurate and reproducible placement of heart valve implants.

EXAMPLE 1 Assay for α-Gal Epitopes' Elimination from Heart valve byα-Galactosidase

In this example, an ELISA assay for assessing the elimination of α-galepitopes from heart valve is conducted.

A monoclonal anti-Gal antibody (designated M86) which is highly specificfor α-gal epitopes on glycoproteins is produced by fusion of splenocytesfrom anti-Gal producing knock-out mice for α 1,3 galactosyltransferase,and a mouse hybridoma fusion partner.

M86 binds to synthetic α-gal epitopes linked to bovine serum albumin(BSA), to bovine thyroglobulin which has 11 α-gal epitopes, R. G. Spiroet al., Occurrence of α-D-galactosyl residues in the thyroglobulin fromseveral species. Localization in the saccharide chains of complexcarbohydrates, 259 J. Biol. Chem. 9858 (1984); or to mouse laminin whichhas 50 α-gal epitopes, R. G. Arumugham et al., Structure of theasparagine-linked sugar chains of laminin. 883 Biochem. Biophys. Acta112 (1986); but not to human thyroglobulin or human laminin, Galβ1-4GlcNAc-BSA (N-acetyllactosamine-BSA) and Galα1-4Galβ1-4GlcNAc-BSA (P1antigen linked to BSA), all of which completely lack α-gal epitopes.Binding is measured at different dilutions of the M86 tissue culturemedium.

Once the M86 antibody is isolated, the monoclonal antibody is dilutedfrom about 1:20 to about 1:160, and preferably diluted from about 1:50to about 1:130. The antibody is incubated for a predetermined period oftime ranging between about 5 hr to about 24 hr, at a predeterminedtemperature ranging from about 3° C. to about 8° C. The antibody ismaintained in constant rotation with fragments of heart valve xenograftmaterial about 5 μm to about 100 μm in size, and more preferably withheart valve fragments ranging from about 10 μm to about 50 μm in size,at various heart valve xenograft material concentrations ranging fromabout 200 mg/ml to about 1.5 mg/ml. Subsequently, the xenograftfragments are removed by centrifugation at centrifugation rate rangingfrom about 20,000×g to about 50,000×g. The proportion of M86 bound tothe xenograft is assessed by measuring the remaining M86 activity in thesupernatant, in ELISA with α-gal-BSA as described in the prior art in,for example, U. Galili et al., Porcine and bovine cartilage transplantsin cynomolgus monkey: II Changes in anti-Gal response during chronicrejection, 63 Transplantation 645-651 (1997). The extent of binding ofM86 to the heart valve material is defined as a percentage inhibition ofsubsequent binding to α-gal-BSA. There is a direct relationship betweenthe amount of α-gal epitopes in the heart valve material and theproportion of M86 complexed with the heart valve material fragments,thus removed from the supernatant (i.e., percentage inhibition).

To perform the assay, fragments of homogenized heart valve xenografttreated with α-galactosidase are incubated with the M86 monoclonalantibody (diluted 1:100) for 20 hr at 4° C. Subsequently, the xengrafttissue fragments are removed by centrifugation at 35,000×g and theremaining M86 in the supernatant is assessed in ELISA with α-gal-BSA assolid phase antigen. The heart valve xenograft tissue is treated with200 U/ml α-galactosidase for 4 hour at 30° C. followed by five washeswith phosphate-buffered solution (PBS), which completely eliminates theα-gal epitopes.

EXAMPLE 2 Assessment of Primate Response to Implanted Porcine HeartValve Xenografts Treated with α-Galactosidase

In this example, porcine peritoneum heart valve implants are treatedwith α-galactosidase to eliminate α-galactosyl epitopes, the implantsare transplanted into cynomolgus monkeys, and the primate response tothe heart valve implants is assessed.

Porcine peritoneum is harvested for forming heart valve xenografts andadherent fatty and/or muscular tissues surgically removed. The specimensare washed for at least five minutes with an alcohol, such as ethanol orisopropanol, to remove lipid soluble contaminants. The heart valvespecimens are frozen at a temperature ranging from about −35° C. toabout −90° C., and preferably at a temperature up to about −70° C., todisrupt, that, is to kill, the specimens' fibrochondrocytes.

Each xenograft specimen is cut into two portions. Each first portion isimmersed in a buffer solution containing α-galactosidase at apredetermined concentration. The specimens are allowed to incubate inthe buffer solutions for a predetermined time period at a predeterminedtemperature. Each second portion is incubated under similar conditionsas the corresponding first portion in a buffer solution in the absenceof α-galactosidase and serves as the control.

At the end of the incubation, the heart valve xenograft specimens arewashed under conditions which allow the enzyme to diffuse out. Assaysare performed to confirm the complete removal of the α-gal epitopes.

The porcine peritoneum is formed into heart valves and the heart valvexenograft specimens are implanted in the six cynomolgus monkeysaccording to heart valve implantation procedures known to those ofordinary skill in the art.

The implantation procedures are performed under sterile surgicaltechnique, and the wounds are closed with 3-0 vicryl or a suitableequivalent known to those of ordinary skill in the art. The animals arepermitted unrestricted cage activity and monitored for any sign ofdiscomfort, swelling, infection, or rejection. Blood samples (e.g., 2ml) are drawn periodically (e.g., every two weeks) for monitoring ofantibodies.

The occurrence of an immune response against the xenograft is assessedby determining anti-Gal and non-anti-Gal anti-soft tissue antibodies(i.e., antibodies binding to soft tissue antigens other than the α-galepitopes) in serum samples from the transplanted monkeys. At least twoblood samples are drawn from the transplanted monkeys on the day ofimplant surgery and at periodic (e.g., two week) intervalspost-transplantation. The blood samples are centrifuged and the serumsamples are frozen and evaluated for the anti-Gal and other non-anti-Galanti-soft tissue antibody activity.

Anti-Gal activity is determined in the serum samples in ELISA withα-gal-BSA as solid phase antigen, according to methods known in theprior art, such as, for example, the methods described in Galili et al.,Porcine and bovine cartilage transplants in cynomolgus monkey: II.Changes in anti-Gal response during chronic rejection, 63Transplantation 645-651 (1997).

Assays are conducted to determine whether α-galactosidase treatedxenografts induce the formation of anti-soft tissue antibodies. Formeasuring anti-soft tissue antibody activity, ELISA assays are performedaccording to methods known in the prior art, such as, for example, themethods described in K. R. Stone et al., Porcine and bovine cartilagetransplants in cynomolgus monkey: I. A model for chronic xenograftrejection, 63 Transplantation 640-645 (1997).

The heart valve xenograft specimens are optionally explanted at one totwo months post-transplantation, sectioned and stained for histologicalevaluation of inflammatory infiltrates. Post-transplantation changes inanti-Gal and other anti-soft tissue antibody activities are correlatedwith the inflammatory histologic characteristics (i.e., granulocytes ormononuclear cell infiltrates) within the explanted heart valve, one totwo months post-transplantation, using methods known in the art, as, forexample, the methods described in K. R. Stone et al., Porcine and bovinecartilage transplants in cynomolgus monkey: I. A model for chronicxenograft rejection, 63 Transplantation 640-645 (1997).

Where the heart valve is explanted, the heart valve xenografts areaseptically harvested, using anesthetic procedure, removal of theimplants and closure of the soft tissues (where the animals are allowedto recover). At the time of the xenograft removal, fluid, if present inamounts sufficient to aspirate, is collected from the for possibleimmunologic testing if the gross and histopathologic evaluation of thetransplants indicate good performance of the transplanted heart valvexenograft material.

The animals which have had xenograft implantations are allowed torecover and are monitored closely until the incisions have healed andthe gait is normal. The xenograft samples are collected, processed, andexamined microscopically.

Portions of the heart valve implants and surrounding tissues are frozenin embedding mediums for frozen tissue specimens in embedding molds forimmunohistochemistry evaluation according to the methods known in theprior art. “TISSUE-TEK®” O.C.T. compound which includes about 10% w/wpolyvinyl alcohol, about 4% w/w polyethylene glycol, and about 86% w/wnonreactive ingredients, and is manufactured by Sakura FinTek, Torrence,Calif., is a non-limiting example of a possible embedding medium for usewith the present invention. Other embedding mediums known to those ofordinary skill in the art may also be used. The remaining implant andsurrounding tissue is collected in 10% neutral buffered formalin forhistopathologic examination.

EXAMPLE 3 Assessment of Primate Response to Implanted Heart ValveXenografts Subjected to Freeze Thaw Cycling and Treatment withProteoglycan-Depleting Factors

In this example, porcine peritoneum soft tissue heart valve implants areprepared and frozen to disrupt, that is, to kill the specimens' cells,as described above in Example 2. The heart valve implants are furthertreated with proteoglycan-depleting factors to eliminate substantiallythe proteoglycans from the xenograft. Subsequently, the xenografts aretreated with glycosidase to remove substantially remaining α-galepitopes from the xenograft, as described in Example 2. Substantialelimination of the proteoglycans and the remaining α-gal epitopesinterferes with the ability of the recipient subject's immune system torecognize the xenograft as foreign. The heart valve implants aretransplanted into cynomologous monkeys, and the primate response to theheart valve implants is assessed.

Heart valve implants from porcine peritoneum are prepared following thepreparation procedures outlined in Example 2 including thesterilization, and freeze/thaw cycling treatments. A chondroitinase ABCsolution is then prepared by combining 0.05M Tris-HCL (7.88gm/liter−MW=157.60), 5 mM benzamidine-HCL (0.783 gm/liter−MW=156.61),0.010 M N-ethylmaleimide (1.2513 gm/liter−MW=125.13), and 0.001Mphenylmethylsulfonyl fluoride (0.17420 gm/liter−MW=174.2), dissolved inmethanol. A mixture of 0.15 M NaCl (8.775 gm/liter−MW=58.5), penicillinand streptomycin (1% (v/v) 10 ml/liter) along with enzyme in the amountof 1 unit chondroitinase ABC (Sigma #C-3509) Enzyme Solution per 1 ml ofsolution is added to bring the solution to 1 liter.

Each heart valve xenograft specimen is incubated in the chondroitinaseABC enzyme solution at a concentration of 1 ml of solution per a 3 mmdiameter heart valve plug. The incubations are performed at a pH of 8.0and 37 degrees C. in a shaker water bath for 48 hours. After theincubation, each heart valve specimen is washed in appropriate bufferand the washings are added to the chondroitinase ABC solution. Eachheart valve specimen is then re-incubated with the chondroitinase ABCsolution at a concentration of 1 unit chondroitinase ABC (Sigma #C-3509)Enzyme Solution per 1 ml of solution for another 48 hours as describedabove. Each heart valve specimen is again washed in appropriate buffersolution, and the washings are added to the chondroitinase ABC solution.

Each heart valve specimen is then incubated in 1 ml of trypsin solution(1 mg/ml trypsin, 0.15 M NaCl, 0.05 M Na Phosphate) at a pH of 7.2 for24 hours. The incubation is performed in a shaker water bath at 37degrees C. Each heart valve specimen is washed in appropriate buffersolution, and the washings are added to the trypsin solution.

Each specimen is then placed in 1 ml of hyaluronidase solution (0.01mg/ml testicular hyaluronidase, 0.005 M Benzamidine HCL, 001 M PMSF,0.010M Nethylmaleimide, 0.005 M Benzamidine HCL, 1% v/v penicillin andstreptomycin) at a pH 6.0 for 24 hours. The incubation is performed in ashaker water bath at 37 degrees C. Each heart valve specimen is thenrinsed again in an appropriate buffer solution, and the washings areadded to the hyaluronidase solution.

Subsequently, the implants are treated with glycosidase as describedabove in Example 2, implanted into the monkeys, and the occurrence of animmune response against each of the xenografts is assessed as describedabove in Example 2.

Those of skill in the art will recognize that the invention may beembodied in other specific forms without departing from the spirit oressential characteristics thereof. The presently described embodimentsare therefore to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description, and all variations ofthe invention which are encompassed within the meaning and range ofequivalency of the claims are therefor intended to be embraced therein.

EXAMPLE 3 Assessment of Primate Response to Implanted Heart ValveXenografts Subjected to Freeze Thaw Cycling and Treatment withProteoglycan-Depleting Factors

In this example, porcine peritoneum soft tissue heart valve implants areprepared and frozen to disrupt, that is, to kill the specimens' cells,as described above in Example 2. The heart valve implants are furthertreated with proteoglycan-depleting factors to eliminate substantiallythe proteoglycans from the xenograft. Subsequently, the xenografts aretreated with glycosidase to remove substantially remaining α-galepitopes from the xenograft, as described in Example 2. Substantialelimination of the proteoglycans and the remaining α-gal epitopesinterferes with the ability of the recipient subject's immune system torecognize the xenograft as foreign. The heart valve implants aretransplanted into cynomologous monkeys, and the primate response to theheart valve implants is assessed.

Heart valve implants from porcine peritoneum are prepared following thepreparation procedures outlined in Example 2 including thesterilization, and freeze/thaw cycling treatments. A chondroitinase ABCsolution is then prepared by combining 0.05M Tris-HCL (7.88gm/liter−MW=157.60), 5 mM benzamidine-HCL (0.783 gm/liter−MW=156.61),0.010 M N-ethylmaleimide (1.2513 gm/liter−MW=125.13), and 0.001 Mphenylmethylsulfonyl fluoride (0.17420 gm/liter−MW=174.2), dissolved inmethanol. A mixture of 0.15 M NaCl (8.775 gm/liter−MW=58.5), penicillinand streptomycin (1% (v/v) 10 ml/liter) along with enzyme in the amountof 1 unit chondroitinase ABC (Sigma #C-3509) Enzyme Solution per 1 ml ofsolution is added to bring the solution to 1 liter.

Each heart valve xenograft specimen is incubated in the chondroitinaseABC enzyme solution at a concentration of 1 ml of solution per a 3 mmdiameter heart valve plug. The incubations are performed at a pH of 8.0and 37 degrees C. in a shaker water bath for 48 hours. After theincubation, each heart valve specimen is washed in appropriate bufferand the washings are added to the chondroitinase ABC solution. Eachheart valve specimen is then re-incubated with the chondroitinase ABCsolution at a concentration of 1 unit chondroitinase ABC (Sigma #C-3509)Enzyme Solution per 1 ml of solution for another 48 hours as describedabove. Each heart valve specimen is again washed in appropriate buffersolution, and the washings are added to the chondroitinase ABC solution.

Each heart valve specimen is then incubated in 1 ml of trypsin solution(1 mg/ml trypsin, 0.15 M NaCl, 0.05 M Na Phosphate) at a pH of 7.2 for24 hours. The incubation is performed in a shaker water bath at 37degrees C. Each heart valve specimen is washed in appropriate buffersolution, and the washings are added to the trypsin solution.

Each specimen is then placed in 1 ml of hyaluronidase solution (0.01mg/ml testicular hyaluronidase, 0.005 M Benzamidine HCL, 001 M PMSF,0.010M Nethylmaleimide, 0.005 M Benzamidine HCL, 1% v/v penicillin andstreptomycin) at a pH 6.0 for 24 hours. The incubation is performed in ashaker water bath at 37 degrees C. Each heart valve specimen is thenrinsed again in an appropriate buffer solution, and the washings areadded to the hyaluronidase solution.

Subsequently, the implants are treated with glycosidase as describedabove in Example 2, implanted into the monkeys, and the occurrence of animmune response against each of the xenografts is assessed as describedabove in Example 2.

Those of skill in the art will recognize that the invention may beembodied in other specific forms without departing from the spirit oressential characteristics thereof. The presently described embodimentsare therefore to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description, and all variations ofthe invention which are encompassed within the meaning and range ofequivalency of the claims are therefor intended to be embraced therein.

1. A heart valve xenograft for implantation into a human comprising aportion of a naturally occurring porcine heart valve, wherein theportion includes a plurality of extracellular components, a plurality ofsubstantially only dead cells, the extracellular components havingreduced proteoglycans, and a plurality of crosslinked proteins of theextracellular components, the dead cells and extracellular componentshaving substantially no surface carbohydrate moieties that aresusceptible to glycosidase digestion, and whereby the portion of theheart valve is substantially non-immunogenic and has substantially thesame mechanical properties as a native heart valve.
 2. The heart valvexenograft according to claim 1, wherein the extracellular components andthe substantially only dead cells have substantially no surfacealpha-galactosyl moieties.
 3. The heart valve xenograft according toclaim 1, wherein the crosslinked proteins are aldehyde-crosslinked. 4.The heart valve xenograft according to claim 3, wherein the aldehyde isglutaraldehyde.
 5. A heart valve xenograft for implantation into a humancomprising a portion of a naturally occurring porcine heart valve,wherein the portion includes a plurality of extracellular components, aplurality of substantially only dead cells, and a plurality ofcrosslinked proteins of the extracellular components, the dead cells andextracellular components having substantially no surface carbohydratemoieties that are susceptible to glycosidase digestion, and whereby theportion of the heart valve is substantially non-immunogenic and hassubstantially the same mechanical properties as a native heart valve. 6.A heart valve xenograft for implantation into a human comprising aportion of a naturally occurring porcine heart valve, wherein theportion includes a plurality of extracellular components, a plurality ofsubstantially only dead cells, the dead cells and extracellularcomponents having substantially no surface carbohydrate moieties thatare susceptible to glycosidase digestion, and whereby the portion of theheart valve is substantially non-immunogenic and has substantially thesame mechanical properties as a native heart valve.
 7. A heart valvexenograft intermediate product comprising a portion of a naturallyoccurring porcine heart valve, wherein the portion includes a pluralityof extracellular components, a plurality of substantially only deadcells, the extracellular components having reduced proteoglycans, and analdehyde in a concentration ranging from about 0.01 percent to about 5percent crosslinking a plurality of proteins of the extracellularcomponents, the dead cells and extracellular components havingsubstantially no surface carbohydrate moieties that are susceptible toglycosidase digestion, and whereby the portion of the heart valve issubstantially non-immunogenic and has substantially the same mechanicalproperties as a native heart valve.